1. Field of the Invention
This invention relates to an electrophoretic method utilizing an electrophoresis medium membrane containing an aqueous poly(meth)acrylamide gel for determination of base sequences of nucleic acids such as a DNA and a RNA.
2. Background of the Invention
With the rapid advances made in research on genetic engineering in recent years, there has arisen a need for quick operations for determination of base sequences of nucleic acids such as a DNA and a RNA.
In the technique for determining base sequences of nucleic acids, slab electrophoresis using an electrophoresis medium membrane containing an aqueous polyacrylamide gel (hereinafter referred to as a polyacrylamide gel membrane or simply as a gel membrane) is indispensable.
The Maxam-Gilvert method according to a chemical degradation process and the dideoxy method according to an enzyme process have heretofore been utilized as the method of preparing a nucleic acid fragment for base sequence reading or determination of a nucleic acid. The dideoxy method enables determination of many base sequences by a single degradation operation, and is therefore popular.
By way of example, the operation of electrophoresis utilizing the polyacrylamide gel membrane as the electrophoresis medium is carried out in the manner as described below.
A polyacrylamide gel membrane having sample slots (i.e. sample spotting holes) at an upper edge is provided between light-permeable, water-impermeable supports, for example, glass plates or organic polymer sheets formed of polyethylene terephthalate or the like, and is disposed vertically. A predetermined amount of a sample (for example, a .sup.32 P-labeled DNA degradated according to the Maxam-Gilvert method) containing nucleic acid fragments having different numbers of bases is spotted into the sample slots, and is subjected to electrophoresis by the application of an electric field. The electrophoresis is carried out by applying a DC voltage, which gives a predetermined electric field per unit length (for example, within the range of 25 V/cm to 70 V/cm) to the polyacrylamide gel membrane, along a predetermined direction (in general, such that the lower side is at higher potential and the upper side is at lower potential). As long as the electric field is applied, DNA molecules (DNA fragments) as high molecular electrolytes having negative charges migrate from the lower-potential side (cathode) to the higher-potential side (anode). As the DNA molecules migrate, they are separated in the polyacrylamide gel matrix based on differences in their number of bases, i.e. in their molecular weights. After the electrophoresis has been carried out for a predetermined time, the gel membrane is removed from the supports, covered by a thin polymer film, and subjected to autoradiography or processing in a computed radiography apparatus provided with an imaging plate, thereby to form a visible image of the separation pattern. In the case where the electrophoretic separation is carried out by use of a sample containing nucleic acid fragments labeled with a dye or a fluorescent dye, the separation pattern can be converted into a visible image by use of an apparatus provided with a photoelectric conversion element.
With the autoradiography, an X-ray photographic film is closely contacted with the thin film covering the polyacrylamide gel membrane, and exposed to .beta.-rays from .sup.32 P or .sup.35 S at a low temperature (for example, 80.degree. C. below the freezing point) for a predetermined time (for example, within the range of approximately 10 hours to approximately 20 hours). After the exposure, the X-ray photographic film is developed to form a visible image of the electrophoretic separation pattern of the DNA fragments.
The base sequence reading or determination of a DNA can be achieved based on the electrophoretic separation pattern of the DNA fragments obtained in the manner as mentioned above.
However, in the case of the electrophoretic separation pattern of the DNA fragments which is obtained by the conventional method, the band intervals of the separation pattern become markedly wide in the low molecular region wherein the number of bases of the DNA molecule is smaller than approximately 5.times.10.sup.1 (the molecular weight is smaller than approximately 1.5.times.10.sup.4), and become markedly narrow in the high molecular region wherein the number of bases of the DNA molecule is larger than approximately 2.times.10.sup.2 (the molecular weight is larger than approximately 6.times.10.sup.4). In the low molecular region, there arises portions where no electrophoretic separation pattern appears in the gel medium membrane. On the other hand, in the high molecular region, it is difficult to determine the base sequence. Therefore, the number of bases for which the base sequence can be determined with a single electrophoresis medium membrane is limited to a small number.
In order to increase the number of base sequences of a DNA readable or determinable by a single operation of electrophoresis, there has heretofore been employed a method wherein the length of the gel membrane is increased to a value within the range of, for example, 80 cm to 100 cm, a method wherein the DNA is labeled with .sup.35 S, instead of .sup.32 P, in order to increase the intensity of .beta.-rays for the purpose of improving the resolution of the separation pattern, or a method wherein a polyacrylamide gel membrane provided with a gel concentration gradient is employed for the purpose of changing the separation characteristics of the gel membrane. However, even with these improvement methods, large nonuniformity in the band intervals of the separation pattern arises over the range from the low molecular region to the high molecular region of the DNA.
As the technique of controlling the fractionation pattern (band intervals), there has heretofore been known to employ a gel membrane provided with a gel concentration gradient, a membrane provided with a pH buffer concentration gradient, or a membrane provided with a membrane thickness gradient. However, there has not yet been known such a technique that can change the fractionation pattern (band intervals) by controlling the conditions of electrophoresis.